Cloning and sequencing of the endo-cellulase cDNA from Robillarda sp. Y-20.
An endo-cellulase cDNA has been screened from a Robillarda sp. Y-20 cDNA expression library using polyclonal antibodies (immunoscreening). Western blot analysis showed that recombinant CMCase I fused to beta-galactosidase with the molecular weight of approximately 150,000 was expressed in Escherichia coli Y1090. Sequence analysis of the cloned cDNA revealed that it consisted of 1,136 nucleotides. By comparison of the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of the purified protein (residues 1 to 18, determined by protein sequencing), the cDNA was found to lack 44 nucleotides at its 5' end corresponding to residues 1 to 15. Therefore, the mature cellulase (CMCase I) was deduced to be composed of 375 amino acid residues and the molecular weight of its protein was calculated to be 41,004. Yaguchi et al. reported that the N-terminal amino acid sequence of an endo-beta-1,4-glucanase (endo-cellulase) from Schizophyllum commune was homologous with the active site of various hen egg-white type lysozymes, and the homology offered evidence for a lysozyme-type mechanism in enzymatic hydrolysis of cellulase [Biochem. Biophys. Res. Commun. (1983) 116, 408-411]. Pentillä et al. also pointed out that some amino acid homology was found between endo-glucanase I from Trichoderma reesei and the lysozyme of the phage T4 [Gene (1986) 45, 253-263]. From the result of sequence alignment of the endo-cellulase from Robillarda sp. Y-20 and four kinds of lysozymes, there was a possibility that the endo-cellulase from Robillarda sp. Y-20 also hydrolyzes carboxymethyl cellulose by a lysozyme-type mechanism.
Yoshigi
N
N
Pharmaceutical Research Laboratories, Sapporo Breweries Ltd., Shizuoka.
Taniguchi
H
H
Sasaki
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D005658:Fungi / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004247:DNA; D002480:Cellulase